Throughout life, blood cells are continually produced from hematopoietic stem cells (HSCs), which are defined by their multilineage differentiation and self-renewal capacities. HSCs transition through a hierarchy during differentiation, n which the loss of self-renewal is accompanied by a gradual lineage commitment with the acquisition of lineage-specific markers. Bone marrow transplantation (BMT) assays assess HSC functionality by measuring its ability in reconstituting all hematopoietic lineages in lethally irradiated mice. HSCs can be prospectively isolated using cell surface markers. We observe a paradoxical population that co-expresses a mature myeloid -associated marker, Mac1, together with classical HSC markers (Mac1hi HSCs). Preliminary data show that Mac1hi HSCs have similar multi-lineage reconstitution potential to that of classical HSCs. Importantly, Mac1hi HSCs are distinct from classicl HSCs in that they exhibit increased size and granularity and co-expression of another myeloid marker, Gr1. I found that, in contrast to classical LSKs, the frequency of Mac1hi HSCs is not significantly altered in genetic mouse models that manipulate a central HSC signaling axis. These data strongly suggest that Mac1hi HSCs are maintained by novel alternative signaling mechanisms. Therefore, the goal of this proposal is to study the properties, physiological functions, and signal transduction of the novel Mac1hi HSCs in comparison to classical HSCs. Based on this goal, I will pursue two specific aims. Specific Aim 1: Validate and further characterize Mac1hi HSCs. I will determine mRNA and protein expression of Mac1 and Gr1 at the single cell level in Mac1hi HSCs. I will investigate whether Mac1hi HC can be further enriched using recently described combinatorial surface markers: CD34-Flt3- or CD150+CD48-. I will then determine the repopulating frequency and lineage reconstitution of Mac1hi HSC using BMT and examine the self-renewal ability of Mac1hi HSC using secondary BMT. Specific Aim 2: Investigate physiological roles and signaling pathways of Mac1hi HSCs. I will evaluate several facets of HSC properties, including cell cycle, quiescence, and homing abilities, in Mac1hi HSCs. I will examine the role of Mac1 in HSC functions using Mac1-/- mice. Furthermore, I will explore signaling mechanisms in Mac1hi HSCs using knockout mouse models with deregulated cytokine signaling important for classical HSCs. Moreover, to discover potential alternate signaling pathways in Mac1hi HSCs, I will perform genome-wide expression profiling of Mac1hi and classical HSCs. This application studies a novel HSC population, which, to our knowledge, is the first instance of an HSC outside the classical compartment. The findings of this study will likely provide insights that will elucidate basic mechanisms of HSC homeostasis and hematologic disorders with dysregulated HSCs including myeloproliferative disorders and leukemias.